SUMMARY High-risk types of human papillomaviruses (HPV) are responsible for virtually all cases of human cervical carcinoma, as well as an increasing number of other malignancies including those of the head and neck, anus and vulva. Unfortunately, good treatment options for late-stage HPV+ malignancies are not currently available, in large part because the virus encodes a protein, E6, which disables cellular apoptotic pathways by accelerating the degradation of molecules such as FADD, caspase 8 and p53. This makes it difficult to eliminate HPV+ cancer cells using conventional inducers of apoptosis. To overcome this obstacle, our laboratory has identified several small molecules that block the binding between E6 and partners such as caspase 8 and E6AP. According to our working model, the use of such molecules in a combinatorial manner will greatly increase the effectiveness of standard radio- and chemotherapeutic treatments. Both in vitro binding data and cellular data from our laboratory provide strong data in support of this working model, and it is now time to test our approach in an in vivo context. The overall objective of this current application, therefore, is to move our exciting in vitro and cellular observations into a mouse xenograft model. We will do this by combining spinacine, our best molecular candidate, with two potential therapeutic agents: TRAIL, a biologic, and cisplatin, a more conventional chemotherapeutic, asking whether either of these combinations can reduce or eliminate the growth of HPV+ tumors, of either cervical or head and neck origin, in a nude mouse model. In particular, we will: 1) Determine the toxicity of spinacine in mice. We will assess the toxicity of spinacine in mice, defining the maximum tolerated dose and identifying the optimum dose with which to carry out experiments designed to test its efficacy, and 2) Evaluate the ability of spinacine to synergize with TRAIL- and/or chemo-based therapies to reduce or eliminate HPV+ tumor growth. We will assess the ability of spinacine to synergize with hrTRAIL and/or the DNA damaging drug cisplatin to inhibit tumor growth in a xenograft model. At the conclusion of this work, we will have 1) Determined the toxicity of the E6-inhibiting molecule spinacine in mice, and 2) Evaluated the effectiveness of combining spinacine with TRAIL- and cisplatin-based treatments in an animal model. This work has the potential to save the lives of thousands of patients suffering from HPV-associated malignancies.